These studies were conducted to investigate the mechanism by which insoluble indium compounds cause pulmonary toxicity and pleural fibrosis in male B6C3F1 mice. Insoluble particles deposited in the respiratory tract are typically cleared by ciliary movement and /or removed by macrophages. We hypothesized that phagosome acidification within macrophages after particle uptake results in the solubilization of the InP and ITO particles, which generates free indium metal ions, the cytotoxic entity. In this study, we first characterized the solubility of InP and ITO particles and observed that both were soluble at acidic pH (pH 4) by ICP-MS. We then characterized the in vitro cytotoxicity of InP and ITO particles on macrophages and lung epithelial cells. The mouse macrophage cell line RAW 264.7 was treated with InP or ITO for 1.5 hr to allow particle uptake. The cells were then rinsed with media to remove extracellular particles and cultured for 24 hr. Cytotoxicity was measured after 24 hr using the MTT viability and LDH assays. InP and ITO-treated RAW 264.7 cells exhibited increased cell death relative to media-treated controls. Similar cytotoxic effects were observed after treatment and uptake of particles by BAL-derived primary mouse alveolar macrophages. Pre-treatment of RAW 264.7 cells for 30 min with 25 nM bafilomycin A1, a specific inhibitor of phagosome acidification, followed by treatment for 1.5 hr with InP (100 g/ml) or ITO (300 g/ml) + 25 nM bafilomycin A1 still resulted in particle uptake, but the cytotoxicity of InP and ITO particles was reduced. The mouse lung epithelial cell line LA-4 was also treated with InP or ITO particles;however, neither particle was cytotoxic to LA-4 cells despite being phagocytosed. These data indicate that phagosome acidification after particle uptake by macrophages in vitro is required for the macrophage cytotoxicity of InP and ITO metal particles. These results also support the hypothesis that particulate indium compounds require solubilization in order to be toxic.